remaining foci co-localizing with the ER [11]. Finally, one can
immunostain ribosomes, which are partially removed by digitonin extraction, with the extraction-resistant signal
co-localizing with the ER [16].
19. This buffer is optimized for Stellaris mRNA probes, and the
concentration of formamide may vary with different FISH
probes used. These washes should be performed with 500 μL
droplets (see Subheading 3.2, steps 2 and 9) to conserve on
formamide and reduce waste.
20. If the back surface of the coverslip is wet, it may draw the wash
or the FISH hybridization buffer up over the top of the coverslip and away from the cell-facing side. This will lead to ineffective washing and staining. Dried salt crystals on the back
surface will also cause problems. These salt crystals can be
removed using a Kimwipe that has been moistened with some
water.
21. All experiments should include a coverslip where the Stellaris
probes are omitted (an example is shown in Fig. 1), or even
better, with a set of probes with scrambled sequences. Ideally,
cells that do not express the mRNA of interest (e.g., due to
CRISPR/Cas9 deletion of its gene) make the best controls.
Images taken of these cells will give a baseline of the type of
fluorescence one should expect due to autofluorescence and
binding of probes to nonoptimal target sequences. This will
give some degree of certainty that the foci observed in a stained
sample are brighter than either autofluorescence or probes
hybridizing to off-target mRNAs. Despite this, unstained
cells can have foci-resembling smFISH signals (see example in
Fig. 1g). These controls are especially important when one is
analyzing mRNAs stained with few probes, such as the
Sec61β mRNA.
22. Provided that the back side of the coverslip is dry and does not
have any residual salt crystals, the liquid should seep underneath it by capillary action. If too much of the FISH hybridization buffer has evaporated, the wash buffer may not effectively
seep underneath the coverslip. In this case, use a pair of forceps
to slowly pry the coverslip from the parafilm until the liquid is
drawn under. If this procedure is done too quickly, the added
sheer force may disrupt the integrity of the cells. Also, do not
use excessive force, as this may break the coverslip. If this is a
constant problem, consider using more probe solution so that
it does not dry over the course of incubation. Alternatively, you
can place a stack of Kimwipes drenched in deionized water in
the incubation chamber to increase the humidity.
23. Co-staining of mRNA by smFISH with an ER marker by
immunofluorescence can help to assess the degree of ER
Visualizing ER-Associated mRNA
47
immunostain ribosomes, which are partially removed by digitonin extraction, with the extraction-resistant signal
co-localizing with the ER [16].
19. This buffer is optimized for Stellaris mRNA probes, and the
concentration of formamide may vary with different FISH
probes used. These washes should be performed with 500 μL
droplets (see Subheading 3.2, steps 2 and 9) to conserve on
formamide and reduce waste.
20. If the back surface of the coverslip is wet, it may draw the wash
or the FISH hybridization buffer up over the top of the coverslip and away from the cell-facing side. This will lead to ineffective washing and staining. Dried salt crystals on the back
surface will also cause problems. These salt crystals can be
removed using a Kimwipe that has been moistened with some
water.
21. All experiments should include a coverslip where the Stellaris
probes are omitted (an example is shown in Fig. 1), or even
better, with a set of probes with scrambled sequences. Ideally,
cells that do not express the mRNA of interest (e.g., due to
CRISPR/Cas9 deletion of its gene) make the best controls.
Images taken of these cells will give a baseline of the type of
fluorescence one should expect due to autofluorescence and
binding of probes to nonoptimal target sequences. This will
give some degree of certainty that the foci observed in a stained
sample are brighter than either autofluorescence or probes
hybridizing to off-target mRNAs. Despite this, unstained
cells can have foci-resembling smFISH signals (see example in
Fig. 1g). These controls are especially important when one is
analyzing mRNAs stained with few probes, such as the
Sec61β mRNA.
22. Provided that the back side of the coverslip is dry and does not
have any residual salt crystals, the liquid should seep underneath it by capillary action. If too much of the FISH hybridization buffer has evaporated, the wash buffer may not effectively
seep underneath the coverslip. In this case, use a pair of forceps
to slowly pry the coverslip from the parafilm until the liquid is
drawn under. If this procedure is done too quickly, the added
sheer force may disrupt the integrity of the cells. Also, do not
use excessive force, as this may break the coverslip. If this is a
constant problem, consider using more probe solution so that
it does not dry over the course of incubation. Alternatively, you
can place a stack of Kimwipes drenched in deionized water in
the incubation chamber to increase the humidity.
23. Co-staining of mRNA by smFISH with an ER marker by
immunofluorescence can help to assess the degree of ER
Visualizing ER-Associated mRNA
47